Transgenic pig models of cystic fibrosis

ABSTRACT

This invention relates to transgenic pig models of cystic fibrosis, cells that can be used to make such animals, and methods of making and using these pigs and cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of U.S. Ser. No.61/555,348, filed Nov. 3, 2011, the contents of which are incorporatedherein by reference.

FIELD OF THE INVENTION

This invention relates to transgenic pig models of cystic fibrosis,cells that can be used to make such animals, and methods of making andusing these pigs and cells.

BACKGROUND OF THE INVENTION

Cystic fibrosis (CF) is caused by genetic mutations in the gene encodingthe cystic fibrosis transmembrane conductance regulator (CFTR).Disrupted CFTR function leads to a number of clinical manifestationsincluding intestinal obstruction, pancreatic insufficiency, focalbiliary cirrhosis, infertility, and recurrent and chronic airwayinfections resulting in progressive respiratory disease (Welsh et al.,Cystic Fibrosis. In The Metabolic and Molecular Basis of InheritedDisease, Scriver et al. (eds.), New York, McGraw-Hill, 2001; Rowe etal., N. Engl. J. Med. 352:1992-2001, 2005).

In human CF, approximately 10-20% of infants are born with an intestinalobstruction termed meconium ileus (van der Doef et al., Curr.Gastroenterol. Rep. 13:265-270, 2011). This obstruction, due to failedpassage of meconium, most commonly occurs in the distal ileum andproximal colon and requires either conservative or surgicalinterventions to relieve the obstruction (Rescorla et al., World J.Surg. 17:318-325, 1993; Murshed et al., Eur. J. Pediatr. Surg.7:275-277, 1997). The exact pathogenesis of meconium ileus remains to bedetermined, but likely results, in part, from defective CFTR-mediatedCl⁻ and/or HCO₃ ⁻ transport by the intestinal epithelium (Clarke et al.,J.O.P. 2:263-267, 2001; Harmon et al., Nat. Med. 16:313-318, 2010;Garcia et al., J. Clin. Invest. 119:2613-2622, 2009).

In order to better understand the pathogenesis of CF, CF animal modelshave been developed including CF mice and more recently CF pigs andferrets (Grubb et al., Physiol. Rev. 79:S193-214, 1999; Rogers et al.,Science 321:1837-1841, 2008; Sun et al., J. Clin. Invest. 120:3149-3160,2010). A clinical feature of these animal models is the presence ofintestinal disease (Grubb et al, Am. J. Physiol. 273:C21-29, 1997; Sunet al., J. Clin. Invest. 120:3149-3160, 2010; Rogers et al., Science321:1837-1841, 2008; Meyerholz et al., Am. J. Pathol. 176:1377-1389,2010; Ostedgaard et al., Sci. Transl. Med. 3:74ra24, 2011). Most CF micemodels have an intestinal phenotype. However, the clinical andhistopathological features are more similar to the distal intestinalobstruction syndrome (DIOS) observed in older humans with CF as opposedto meconium ileus in infants with CF. For example, a small fraction ofCF mice die from intestinal disease within days after birth, but mostmortality occurs at the time of weaning. In addition, the intestinalobstruction seems to result from intestinal mucus accumulation asopposed to a failure to pass meconium at birth as observed in human CF(Oppenheimer et al., Bull. Johns Hopkins Hosp. 111:1-13, 192;Wilschanski et al., J. R. Soc. Med. 91 Suppl. 34:40-49, 1998).

We have recently developed a porcine model of CF (Rogers et al., J.Clin. Invest. 118:1571-1577, 2008; Rogers et al., Science 321:1837-1841,2008; Ostedgaard et al., Sci. Transl. Med. 3:74ra24, 2011). CF pigsdisplay many of the same features as humans with CF including meconiumileus, exocrine pancreatic destruction, focal biliary cirrhosis,micro-gallbaldder, vas deferens abnormalities, and airway disease(Rogers et al., Science 321:1837-1841, 2008; Meyerholz et al., Am. J.Pathol. 176:1377-1389, 2010; Stoltz et al., Sci. Transl. Med. 2:29ra31,2010; Ostedgaard et al., Sci. Transl. Med. 3:74ra24, 2011; Welsh et al.,Cystic Fibrosis. In The Metabolic and Molecular Basis of InheritedDisease, Scriver et al. (eds.), New York, McGraw-Hill, 2001; Rowe etal., N. Engl. J. Med. 352:1992-2001, 2005). In contrast to humans, thereis a 100% penetrance of meconium ileus in both CFTR−/− andCFTRΔF508/ΔF508 newborn piglets. Features of the CF pig meconium ileusvery closely replicate that observed in humans with CF includingobstruction by meconium in the distal bowel, atretic intestinalsegments, and microcolon (Meyerholz et al., Am. J. Pathol.176:1377-1389, 2010; Rogers et al., Science 321:1837-1841, 2008;Ostedgaard et al., Sci. Transl. Med. 3:74ra24, 2011). The meconium ileusin CF pigs is lethal if not corrected within 24-36 hours after birth.Limited attempts at non-surgical correction (gastrograffin enemas) ofthe intestinal obstruction in CF pigs have been unsuccessful, in part,due to the atretic intestinal segments. Yet, we have been able tosurgically correct the meconium ileus in both CFTR−/− andCFTRΔF508/ΔF508 pigs (Rogers et al., Science 321:1837-1841, 2008; Stoltzet al., Sci. Transl. Med. 2:29ra31, 2010; Ostedgaard et al., Sci.Transl. Med. 3:74ra24, 2011). Despite the success of surgical correctionof the meconium ileus in CF pigs, the surgical procedure can beassociated with morbidity and mortality and is still not feasible inmost CF pigs due complications associated with meconium ileus includingintestinal atresia and in utero intestinal perforation.

SUMMARY OF THE INVENTION

The invention provides transgenic pigs including a genome that has (i)one or more mutations in both alleles of the pig cystic fibrosistransmembrane regulator (CFTR) gene resulting in altered pig CFTRexpression and one or more symptoms of cystic fibrosis (CF) in the pigs;and (ii) a CFTR transgene (e.g., a wild-type or other functional CFTRtransgene; e.g., a human or porcine gene) under the control of promoterresulting in CFTR expression in intestinal cells of the pigs (e.g., anintestinal cell-specific promoter (e.g., an intestinal fatty acidbinding protein (iFABP) promoter, such as that of a rat)). The pigs ofthe invention exhibit reduced meconium ileus relative to pigs lackingexpression of a CFTR transgene in intestinal cells. As described furtherherein, the pigs of the invention can have one or more phenotypesselected from the group consisting of (a) an electrophysiologicalphenotype similar to that of human cystic fibrosis, (b) pancreaticinsufficiency or abnormalities, (c) hepatic abnormalities, (d) gallbladder and/or bile duct abnormalities, (e) tracheal abnormalities, (f)cystic fibrosis lung disease, (g) sweat gland abnormalities, and (h)kidney abnormalities. In certain examples, the CFTR alleles of thegenomes of pigs have been knocked out and/or replaced with mutant CFTRgenes (e.g., human or porcine genes), the expression of which results ina CF phenotype. In one example, the mutant CFTR gene (e.g., human orporcine gene) includes a deletion of F508.

The invention also provides methods of determining whether a candidatetherapeutic approach can be used in the treatment of cystic fibrosis.These methods involve carrying out a therapeutic approach (e.g.,administering a candidate therapeutic agent) to a transgenic pig asdescribed herein, and monitoring the pig for a symptom of cysticfibrosis. Detection of improvement in a symptom of cystic fibrosisindicates the identification of a therapeutic approach (e.g., acandidate therapeutic agent, such as a candidate compound) that can beused in the treatment of cystic fibrosis. In certain examples, thesymptom of cystic fibrosis is monitored in the lung, pancreas, liver, orkidney of the pig.

Also included in the invention are isolated cells or tissues of the pigsof the invention, homozygous and heterozygous forms of the pigs of theinvention, and isolated cells and tissues thereof. Further, cells usedto make the animals of the invention are included in the invention.

Other features and advantages of the invention will be apparent from thedrawings, the detailed description, the experimental examples, and theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A) Schematic of the transgene vector (10871 bp) with porcineCFTR cDNA (1790-6238) driven by the rat intestinal fatty acid bindingprotein (iFABP) promoter (209-1419), flanked by the intervening sequenceand the bovine growth hormone poly-A (BGHpA) (6295-6508), and followedby a hygromycin cDNA sequence (7392-8415) flanked by SV40 (7049-7373)and SV40 poly-A (8428-8800) signal sequences. Bgl I restriction site isdenoted. (B) Gross image of a meconium plug (green-black colored portionof stool) that was passed by a CFTR−/−; Tg-FABP-pCFTR pig (line 1e)following an enema at approximately 18 h after birth. After the meconiumplug was passed, a transition to normal-appearing stool was observed(yellow-green stool denoted by white arrows) bar=2 em. (C) Gross imagesfrom the gastrointestinal tract of CFTR−/−; Tg-FABP-pCFTR piglets. Atbirth, in 2 of the 5 lines meconium ileus was alleviated in CFTR−/−;Tg-FABP-pCFTR pigs. Meconium ileus lesions ranged from absent (leftpanel, line 1a) with normal sized intestine and colon (black arrow) tosevere meconium obstruction (right panel, line 1c, white asterisks)often located in the spiral colon with distal microcolon (white arrow).Middle panel—Gross image from the intestinal tract of a CFTR−/−;Tg-FABP-pCFTR pig (line 1a) at 6 d of age. The intestine and spiralcolon (arrow) is of normal size and coloration. bar=1.3 cm. (D) CFTRmRNA expression in ileal segments from CFTR+/+, CFTR−/−, and CFTR−/−;TgFABP-pCFTR newborn piglets. CFTR mRNA levels were determined withquantitative RT-PCR (relative to beta-actin) and values are expressed asa percentage of CFTR mRNA levels relative to CFTR+/+ levels. n=2-4 pergenotype. Bars represent mean±SEM. (E) Southern blot of amplifiedgenomic DNA from rat iFABP-pCFTR transgenic pig fetal CFTR−/−fibroblasts. Bann-digested amplified genomic DNA was hybridized with aprobe that detected exon 13 of porcine CFTR. Endogenous CFTR yields a˜2.2 kb band and iFABP-pCFTR ˜3.5 kb. Lanes 1-18 contain DNA fromindividual fibroblast clones. WT well contains DNA from a wild-typecontrol pig. Positive control (+) well contains the linearizediFABP-pCFTR plasmid construct. Clones 1E (lane 2), 1A (lane 4), 1D (lane14), 1C (lane 17), and 1B (lane 18) were selected for somatic cellnuclear transfer into enucleated oocytes.

FIG. 2. (A-E) Representative Isc tracings from excised ileal segmentsmounted in Ussing chambers for electrophysiological studies. (A)CFTR+/+, (B) CFTR−/−, (C) CFTR−/−; Tg-iFABP-pCFTR. Tissue samples wereobtained from newborn piglets. The following agents were addedsequentially: apical 100 μm amiloride (amil), apical 10 forskolin(F)/100 μm IBMX (I), apical 100 μm GlyH, basolateral 100 μm carbachol(CCH), apical 100 μm DIDS, apical 5 mM dextrose (Dex), and apical 200 μmphlorizin (Phz). (D) Changes in Isc following addition of forskolin/IBMXto freshly excised ileal segments from newborn CFTR+/+(n=8), CFTR−/−(n=7), and CFTR−/−; TgFABP-pCFTR (n=1 for 1b, 3 for 1c, 3 for 1d, 4 for1e, and 2 for 1a) piglets. Prior to forskolin/IBMX treatment, tissueswere exposed to amiloride. Bars represent mean±SEM. (E) Ilealshort-circuit current following forskolin and IBMX treatment versusileal CFTR mRNA. All data are shown as a percentage of wild-type levels.Red circles denote cell lines (litters) that had a severe meconium ileusphenotype and green circles denote cell lines (litters) that had areduction in the severity of meconium ileus.

FIG. 3. (A-E) Newborn CFTR−/−; Tg-FABP-pCFTR pig pancreas. (A) Lowpowerimage showing loss of lobularity. (B) High-power image showing variableextent of zymogen staining (dPAS staining) in acinar cells. (C) Grossimages of CFTR−/−; Tg-FABP-pCFTR gallbladders. Gallbladders ranged insize from only a few mm (left panel) to moderate sized (right panel) incross sectional diameter. Bar=7.7 mm. (D) Microscopic images of CFTR−/−;Tg-FABP-pCFTR gallbladders. Gallbladders varied from moderate sized andpatent with focal mucous change in the epithelium to severemicrogallbladder that was obstructed by mucus with diffuse mucinouschange in the epithelium. (E) Microscopic images of livers from CFTR−/−;Tg-FABP-pCFTR newborn pigs. There was minimal evidence (left panel) ofportal change to prominent focal biliary cirrhosis-like changes (rightpanel).

FIG. 4. Microscopic images of trachea from a CFTR−/−; Tg-FABP-pCFTRnewborn pig. The tracheas were irregularly shaped, had cartilage ringdefects in the anterior trachea (left panel—arrow), and areas ofabnormal-appearing airway smooth muscle bundles (right panel—arrows)(Meyerholz et al., Am. J. Resp. Crit. Care Med. 182:1251-1261, 2010).

FIG. 5. (A-C) Representative Isc tracings from excised tracheas mountedin Ussing chambers for electrophysiological studies. Tracheal sampleswere obtained from newborn piglets. The following agents were addedsequentially: apical 100 μm amiloride, apical 100 μm DIDS, apical 100 μmforskolin(FSK)/10 μm IBMX, and apical 10 μm GlyH. (D-F) Changes in Iscfollowing addition of FSK/IBMX to (D) freshly excised tracheal segmentsfrom newborn CFTR+/+(n=10), CFTR−/− (n=8), and CFTR−/−; TgFABP-pCFTR(n=3 for 1b, 2 for 1c, 4 for 1d, 4 for 1e, and 1 for 1a) piglets; (E)cultured tracheal epithelia from newborn CFTR+/+(n=44), CFTR−/− (n=15),and CFTR−/−; TgFABP-pCFTR (n=1 for all cell lines except for 1d in whicha sample was unavailable) piglets; and (F) cultured nasal turbinateepithelia from newborn CFTR+/+(n=*), CFTR−/− (n=*), and CFTR−/−;TgFABP-pCFTR (n=3 for 1b, 1 for 1c, 1 for 1d, 2 for 1e, and 1 for 1a)piglets. Prior to FSK/IBMX treatment, tissues were sequentially exposedto amiloride and DIDS. Bars represent mean±SEM.

FIG. 6. Case #3 (62 d). (A) Low-power microscopic image of the trachea.The trachea is malformed in shape with anterior cartilagenous ringdefects (arrow) and significant overlap of cartilage rings in theposterior trachea. (B) Microscopic images of the pancreas, top panel(2×), bottom panel (20×). Fibroadipose tissue replacement of thepancreas was prominent (top) with dilated ducts (bottom) and acinidetected. (C) others had evidence of severe bridging cirrhosis (rightpanels). (D) Microscopic images of the liver from case #3 (62 d). Atnecropsy, the changes in the liver were heterogeneous. Some regions werenormal-appearing (left panels) and others had evidence of severebridging cirrhosis (right panels). Brown staining is for smooth musclealpha-actin. (E) Microscopic images of the liver from case #3 (62 d). Atnecropsy the changes in the liver were heterogenous. Some regions werenormal-appearing (left panels) and others had evidence of severebridging cirrhosis (right panels). Red picrosirius staining forcollagen.

FIG. 7. Microscopic images of the pancreas from cases #4 (332 d) and #5(435 d). (A) Remnant exocrine pancreas was composed mostly of adiposetissue (asterisks) with sparse remnant pancreas. (B) Dilated cysticducts (arrows). (C) and (D) show remnant pancreas composed of scantexocrine tissue and intact islets (arrows). HE stain. (E-H) Microscopicimages of the gallbladder from cases #4 and #5. (E) There ismucopurulent and necrotic debris in the gallbladder lumen (asterisk). Inthe necrotic areas, there were many (F) cellular foci (G) densecalcifications (black staining) and (H) inflammatory cells (arrow) andluminal mucus (asterisk).

FIG. 8. (A) Microscopic images of the vas deferens from case #5 (435 d).The vas deferens epithelium (arrow, left lower panel), when detected,was often collapsed with eosinophilic material in the lumen that wasdPAS positive (inset) or more often completely absent (right panels).(B) Microscopic images of the vas deferens from case #5 (435 d) withdifferent stains (20×).

FIG. 9. (A) Serial chest X-ray computed tomography images from aCFTR−/−; TgFABP-pCFTR pig (case #5). Airway wall thickening (arrows) waspresent. At 92 d, obstruction of the tracheal bronchus was observed withcollapse of the associated lung segment (asterisks). This persisteduntil 260 d and was resolving by 308 d. Airway wall thickening andparenchymal changes persisted. (B and C) Total number and differentbacterial species recovered in serial tracheal lobe BAL samples from twoCFTR-I−; TgFABP-pCFTR pigs Case #4 (B and C) and Case #5 (D and E). Dataare color-coded to indicate individual species of bacteria. (F and G)Bronchoalveolar lavage (BAL) neutrophil and 11-8 levels in a CFTR−/−;TgFABP-pCFTR pig (case #5). BAL was performed at indicated time pointsand (F) percent neutrophils and (G) 11-8 were determined on recoveredliquid from the tracheal lobe.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides transgenic pig models of cystic fibrosis (CF) inwhich the meconium ileus phenotype of the pigs is reduced or alleviatedby intestinal expression of the cystic fibrosis transmembrane regulator(CFTR). As discussed in detail above, meconium ileus is a conditioncharacterized by intestinal obstruction, which is caused by failedpassage of meconium, the material present in the bowel prior to birth.We have found that meconium ileus has 100% penetrance and is lethal inour transgenic pig CF models if not corrected within 24-36 hours afterbirth. Prior to the present invention, correction has involved surgery,which can be associated with morbidity and mortality, and is quitedifficult in most CF pigs due to complications associated with theircondition. The present invention eliminates the need for surgery, andthus provides pig models of CF in which post-natal care can besimplified. This provides the opportunity for use and study of themodels, without complications associated with surgery.

The pigs of the invention are CF models due to mutation and/orinactivation of both alleles of their endogenous CFTR gene. This isachieved by, for example, knock out of CFTR alleles and/or replacementof CFTR alleles with mutant CFTR sequences (e.g., porcine or human CFTRsequences having, for example, the ΔF508 mutation). The pigs can beproduced using homologous recombination and somatic cell nucleartransfer methods, such as those described in U.S. Pat. No. 7,989,675;US-2009-0241203-A1; Rogers et al., Science 321:1837-1841, 2008; Rogerset al., J. Clin. Invest. 118(4):1571-1577, 2008; Ostedgaard et al., Sci.Transl. Med. 3(74):74ra24, 2011, the teachings of which are incorporatedherein by reference.

According to the invention, the meconium ileus phenotype is corrected oralleviated in CF pig models, such as those described above, byexpression of CFTR (e.g., wild-type CFTR) in intestinal cells of thepig. This can be achieved by use of an intestinal cell-specificpromoter. A construct expressing CFTR can be, for example, introducedinto the genome of a CF model pig by somatic cell nuclear transfer.Preferably, the promoter is specific for intestinal cells so that thecorrection is limited to the intestinal phenotype, including meconiumileus. This was achieved using the iFABP promoter, as described in theexperimental examples set forth below. In addition to this promoter,other promoters can be used including, e.g., the liver FABP promoter andthe villin promoter. Further, in addition to intestinal cell-specificpromoters, those having somewhat broader expression, but still resultingin the maintenance of some CF phenotypes, can be used. The CFTR geneexpressed under the control of such a promoter can be, for example,porcine or human CFTR (e.g., wild-type porcine or human CFTR).

The levels of expression of CFTR in intestinal cells, as well as thespecific cell types in which expression is achieved, can vary, asdetermined to be appropriate by those of skill in the art. Appropriatelevels can be determined by assessment of animals for a desiredphenotype, such as correction or alleviation of meconium ileus. Asgeneral guidance, and based on the experiments described herein,appropriate levels include CFTR expressed at a level greater than 10% ofthat of a comparable sample from a CFTR+/CFTR+ pig, preferably at least20%, 30%, 40%, 50%, 75%, 100%, or more, as determined by analysis ofmRNA expression levels in samples prepared from excised ileal segments,as described below. As additional guidance, appropriate levels includeCFTR expression that leads to more than 27%, preferably 60% or more, ofthat of a comparable sample from a CFTR+/CFTR+ pig, CFTR activity, asdetermined by analysis of forskolin and IBMX-stimulated increasedshort-circuit current in ileal segments, as described below. Also, asexplained further below, the level of intestinal expression of CFTRcorrelates with the intestinal phenotype.

The levels of meconium ileus in pigs of the invention can vary betweenonly a small improvement up to 100% improvement (e.g., 10%, 25%, 50%,75%, 90%, 95% and 99%), as compared with CFTR−/CFTR− and wild-type,CFTR+/CFTR+ animals. The meconium ileus can be assessed by methods knownin the art, such as those described herein, including assessment ofintestinal blockage by direct analysis of tissue or attempted evacuationof any meconium present, or by electrophysiological analysis of affectedtissues, as described herein.

Although the meconium ileus phenotype of the pigs of the invention iscorrected or alleviated, the pigs maintain other phenotypes of cysticfibrosis. Thus, the pigs of the invention can be characterized by one ormore phenotypes including abnormalities in the lungs, other parts of therespiratory tract, pancreas, liver, gall bladder, and vas deferens. Suchabnormalities may include infection and inflammation of any or all ofthe organs noted above, irregularities in tracheal shape and defects intracheal cartilage rings, focal biliary cirrhosis, progressive loss ofexocrine pancreatic tissue and replacement by adipose tissue,gallbladder of small size and including mucus and cellular debris,collapse or absence of the lumen of the vas deferens, andelectrophysiological abnormalities. In more detail, such abnormalitiesmay include one or more of the following:

(i) electrophysiological features similar to CF humans including, e.g.,any one or more of (a) hyperpolarized baseline Vt, (b) reduction of Vtby amiloride, and (c) no CFTR or other Cl⁻ channel activity (as measuredby, e.g., perfusion of apical surface with Cl⁻-free solution andaddition of isoproterenol; perfusion with ATP to activate P2Y2 receptorsand Ca²⁺-activated Cl⁻ channels; and perfusion with the CFTR inhibitorGlyH-101);

(ii) exocrine pancreatic insufficiency or abnormalities, ascharacterized by, e.g., one or more of (a) decreased size, (b)degenerative lobules with, e.g., increased loose adipose and myxomatoustissue, and scattered to moderate cellular inflammation, (c) diminishedeosinophilic zymogen granules in residual acini, (d) variable dilationand obstruction of centroacinar spaces, ductules, and ducts witheosinophilic materal plus infrequent neutrophils and macrophages mixedwith cellular debris, (e) foci of mucinous metaplasia in ducts andductules, and (f) increased redness;

(iii) hepatic abnormalities consistent with focal biliary cirrhosis, ascharacterized by, e.g., any one or more of (a) mild to moderate hepaticlesions, (b) chronic cellular inflammation, (c) ductular hyperplasia,and (d) mild fibrosis;

(iv) gall bladder and/or bile duct abnormalities, as characterized by,e.g., any one or more of (a) gallstones, (b) reduced size, (c) congealedbile and mucus, and (d) epithelia with diffuse mucinous changes withfolds extending into the lumen;

(v) tracheal abnormalities characterized by, for example, altered lumenarea, circumference, submucosal gland area, and smooth muscle area;

(vi) characteristics of CF lung disease, for example, any one or more ofobstruction of some airways with mucopurulent material, scatteredmucopurulent debris in airway luments with chronic purulent to lymphoidairway wall inflammation, surface epithelium with areas of goblet cellhyperplasia, mucocellular material in submucosal glands, lobularatelectasis, host defense defect as shown by detection of a variety ofbacterial species in lung samples; and

(vii) lack of abnormalities in vas deferens and lungs at birth.Additional information concerning the phenotypes of pigs of theinvention is provided below.

The pigs of the invention can be used in screening methods includingthose carried out in the identification and characterization ofapproaches for treating CF. Candidate drugs and other approaches totreatment can therefore be evaluated for effects on CF symptoms in thepigs of the invention. Thus, in addition to the pig models themselves,the invention also provides methods of using the animals for identifyingand characterizing treatments. Further, the invention includes methodsof making the pigs of the invention, as well as cells that can be usedin these methods. Additional details concerning the pig models systems,methods, and cells of the invention are provided as follows. The animalsof the invention can be made using the following general strategy.Briefly, the genome of a cell (e.g., a fetal fibroblast or another cell)from a pig of interest, such as a CFTR−/− pig or a CFTR ΔF508/ΔF508 pig(wherein the mutant CFTR gene is, e.g., porcine or human), isgenetically modified by introduction of one or more CFTR genes under thecontrol of an intestinal cell-specific promoter, preferably bytransfection (e.g., using electroporation) or by gene targeting byhomologous recombination to create a “donor cell.” The nucleus of such agenetically modified donor cell (or the entire donor cell, including thenucleus) is then transferred into a so-called “recipient cell,” such asan enucleated oocyte. After activation and, typically, a brief period ofin vitro culture, the resulting embryo is implanted into a surrogatefemale in which development of the embryo proceeds. Typically, the donorcell, oocyte, and surrogate female are of the same species, but thesources can be different species, as is known in the art. The inventionincludes cells of the pigs of the invention, as well as cells used inmaking the pigs of the invention (e.g., a CFTR−/− or a CFTR ΔF508/ΔF508cell comprising an integrated CFTR transgene under the control of anintestinal cell-specific promoter, as described herein).

Details of methods for making large genetically modified animals, suchas pigs, used in the invention, are provided, for example, in U.S. Pat.No. 7,989,675; US-2009-0241203-A1; Rogers et al., Science 321:1837-1841,2008; Rogers et al., J. Clin. Invest. 118(4):1571-1577, 2008; Ostedgaardet al., Sci. Transl. Med. 3(74):74ra24, 2011. Additional informationconcerning certain aspects of such methods is known in the art and canalso be adapted for use in the present invention (see, e.g., US2005/0120400 A1; U.S. Pat. No. 5,995,577; WO 95/16670; WO 96/07732; WO97/00669; WO 97 00668; WO 2005/104835; Lai et al., Reproductive Biologyand Endocrinology 1:82, 2003; Hao et al., Transgenic Res. 15:739-750,2006; Li et al., Biology of Reproduction 75:226-230, 2006; Lai et al.,Nature Biotechnology 24(4):435-436, 2006; Lai et al., Methods inMolecular Biology 254(2):149-163, 2004; Lai et al., Cloning and StemCells 5(4):233-241, 2003; Park et al., Animal Biotechnology12(2):173-181, 2001; Lai et al., Science 295:1089-1092, 2002; Park etal., Biology of Reproduction 65:1681-1685, 2001; the contents of each ofwhich are incorporated herein by reference).

The transgenic pigs of the invention can be any members of the familySuidae, and in particular any member of the genus Sus, such as Susscrofa, which is also known as the domestic pig or a subspecies thereof(Sus scrofa domestica). In one specific example, the animal is aminiature swine that is a descendent from the miniature swine describedby Sachs et al., Transplantation 22:559, 1976. The pigs of the inventioncan be of any age, including piglets, juvenile pigs, and adult pigs,both female and male.

The transgenic animals of the invention can be used in theidentification and characterization of drug and other treatment methodsfor CF. In these methods, for example, a candidate therapeutic agent canbe administered to an animal and the impact of the agent on a feature ofthe disease exhibited by the animal can be monitored. Optionally, themethods can also involve exposure of the animals to environmental orother conditions known to contribute to or exacerbate the disease orcondition. For example, in the case of CF animal models having impairedrespiratory function, the effect of the drug on such function can beassessed by measurement of standard respiratory parameters. In anotherexample, in the case of animals exhibiting impaired digestion, due toblockage of pancreatic and/or liver ducts, the effect of a treatment ondigestion can be determined.

With the models of the invention, it is possible to test hypotheses thatlead to new treatments and to evaluate potential therapies for CF lungdisease. The models also make it possible to assess electrolytetransport by porcine airway epithelia in vitro and in vivo, the volumeof airway surface liquid in vitro and in vivo, the ion composition ofairway surface liquid in vitro and in vivo, the airway surface liquid pHin the airway, and electrolyte transport in the small airways. It isalso possible to measure respiratory mucociliary transport in vitro andin vivo. For assessing inflammation, several tests and assays can becarried out, including (but not limited to) assays of key markers ofinflammation in amniotic fluid, fetal lung liquid, and bronchoalveolarlavage by using lung tissue histochemistry, large-scale gene expressionprofiling of pulmonary tissues, cytokine and cell assays, andproteomics. It is also possible to raise CF and non-CF piglets inisolators under completely germ free conditions and to test for thedevelopment of pulmonary inflammation, and then selectively expose thepiglets to inflammatory stimuli including bacteria and viruses. Inaddition, investigators can test how loss of CFTR function in airwayepithelia results in altered NFKB signaling, the function of secretedepithelial antimicrobials/host defense proteins, and the consequences ofloss of CFTR function in macrophages or neutrophils. The availability ofthe porcine CF model allows tests of the early manifestations of the CF.The natural history of pulmonary infections in CF pigs can also bemonitored, leading to a determination of whether the airway epithelia ofCF pigs can be colonized by CF or porcine pathogens and/ornon-pathogenic opportunistic organisms.

Although lung disease is the current main cause of mortality, patientssuffer from CF disease in many other organs. Availability of a CF modelallows new investigations and tests of therapeutics in the pancreas,intestine, sweat gland, liver, vas deferens, kidney, and other organsaffected primarily or secondarily by CF. The screening methods of theinvention can be carried out to test the efficacy of new compounds,combinations of new and old compounds, non-pharmaceutical treatments,gene therapy, and combinations of pharmaceutical and non-pharmaceuticaltreatments.

The following Examples are meant to illustrate the invention and are notmeant to limit the scope of the invention in any way.

Experimental Examples Results Generation of CFTR−/−; TgFABP-pCFTR Pigs

We hypothesized that intestinal expression of CFTR in CFTR−/− pigs wouldresult in a milder form of meconium ileus, amenable to correction withconservative techniques or complete correction of the meconium ileusphenotype. We expressed CFTR in the intestinal tract under control ofthe rat iFABP gene promoter (Zhou et al., Science 266:1705-1708, 1994).To produce CFTR−/− gut-corrected pigs (hereafter called CFTR−/−;TgFABP-pCFTR), CFTR−/− fetal fibroblasts (male) were first transfectedwith a rat iFABP-pCFTR construct (FIG. 1A). Antibiotic selection wasperformed followed by confirmation of positive clones with Southernblotting (FIG. 1E). Positive clones were expanded and used as nucleardonors for transfer to enucleated oocytes. Somatic cell nuclear transfer(SCNT) embryos were transferred to surrogate females and piglets weredelivered by cesarean section at term. Five different clones or celllines (1a, 1b, 1c, 1d, and 1e) were transferred individually to a totalof 15 surrogate females (line 1e was transferred to 8 differentsurrogate females). Seven surrogates produced a total of 25 malepiglets. Eight surrogates did not produce offspring.

Transgenic Expression of iFABP-pCFTR Corrects the Meconium IleusPhenotype in CFTR−/− Pigs

One of the goals of this study was to determine if transgenic expressionof CFTR in the intestinal tract, under the control of the iFABPpromoter, would alleviate the meconium ileus in CF pigs. Not knowing ifthis approach would work, we decided to enhance the likelihood ofmeconium ileus correction by treating the majority of animals withenemas after birth. Newborn pigs (21 of the 25 piglets born) received agastrograffin enema shortly after birth and attempts were made to keepthem alive. The other 4 piglets were euthanized shortly after birth forvarious reasons (1 for elective necropsy, 2 for anal malformations, 1for limb malformations).

Within 1 hour of birth, piglets underwent a gastrograffin enema and werehand-fed colostrum containing polyethylene glycol and transitioned topiglet milk replacer. The response to the enema was variable betweenlitters. Following the initial enema, we recovered from the rectumeither nothing, segments of meconium, or small white mucus plugs (˜2 mmin diameter). To enhance removal of meconium from the intestinal tract,repeat gastrograffin enemas were performed at 8-12 h intervals untileither all of the meconium was evacuated (FIG. 1B) or the animals failedto pass meconium despite repeat enemas. The decision to euthanize wasbased upon the lack of a response to enemas (no or minimal meconiumoutput) despite good oral intake.

Of the 5 transgenic lines studied, piglets from 2 lines (1a and 1e) hada favorable intestinal phenotype. FIG. 1C shows images of the intestinaltract from a CFTR−/−; TgFABP-pCFTR newborn piglet (left panel, line 1a)and from a 6 day old CFTR−/−; TgFABP-pCFTR pig (middle panel, line 1a).There was a relatively normal-appearing small intestine, no evidence ofatretic intestinal segments, and a normal-sized spiral colon. Incontrast, piglets from lines 1b, 1c, and 1d had evidence of severemeconium ileus at birth (FIG. 1C, right panel), similar to CFTR−/−newborn piglets in the absence of the transgene. These findings showthat expression of CFTR under the control of the rat iFABP promoter canalleviate/correct the meconium ileus phenotype in CF piglets.

CFTR mRNA Expression Levels Correlate with Intestinal Phenotype

Finding variable levels of meconium ileus correction between theindividual lines suggested that CFTR mRNA expression levels differedbetween CFTR−/−; TgFABP-pCFTR animals from individual clones or celllines. We first performed quantitative RT-PCR for CFTR mRNA on ilealtissue segments from CFTR+/+, CFTR−/−, and CFTR−/−; TgFABP-pCFTR newbornpiglets. Compared to CFTR+/+ ileal samples, CFTR mRNA was essentiallyundetectable in intestinal tissues from CFTR−/− animals (FIG. 1D. In theCFTR−/−; TgFABP-pCFTR animals, we found varying levels of ileal CFTRmRNA expression between the different cell lines used for SCNT (FIG.1D). For example, some lines (1e and 1a) showed high ileal CFTR mRNAlevels, others (1b and 1c) had very low levels of CFTR mRNA in the rangeof those observed in CFTR−/− ileum, and one line had intermediate levels(1d). This variability was not unexpected and is consistent withdiffering transgene insertion sites and/or transgene copy number betweenthe different cell lines used for SCNT.

We next asked if CFTR mRNA levels correlated with intestinal diseasephenotype. CFTR−/−; TgFABP-pCFTR piglets with the highest CFTR mRNAlevels had the least severe meconium ileus and were more likely tosurvive (Table 1). Lines in which the meconium ileus phenotype was notcorrected showed ileal CFTR mRNA levels less than 10% of values fromCFTR+/+ ileum (line 1b=1.9%, line 1c=3.7%, and line 1d 9.4%). Muchhigher levels were observed in the two lines that had a favorableintestinal phenotype. Lines 1e and 1a had ileal CFTR mRNA levels at 21.7and 25.5%, respectively, of levels in CFTR+/+ pigs. These data suggestthat severe meconium ileus occurs with ileal CFTR mRNA levels less thanapproximately 10% of wild-type levels, but that approximately 22% ofwild-type CFTR mRNA expression is sufficient to prevent meconium ileusin CFTR−/− pigs.

TABLE 1 Meconium ileus % wild-type % wild-type Litter PhenotypeCFTR-mRNA ISC_(cAMP) CFTR−/− 3+ 3.5 0 Tg-FABP-pCFTR 1b 3+ 1.9 0Tg-FABP-pCFTR 1c 3+ 3.7 0 Tg-FABP-pCFTR 1d 2+ 9.4 27 Tg-FABP-pCFTR 1e 1+21.7 84 Tg-FABP-pCFTR 1a 1+ 25.5 63 CFTR+/+ 0  100 100Transgenic Expression of iFABP-pCFTR in CFTR−/− Pigs Increases CFTRExpression in Intestinal Crypts

To test for the presence of CFTR protein in intestinal epithelia,immunhistochemistry was performed on ileal samples from CFTR+/+,CFTR−/−, and CFTR−/−; TgFABP-pCFTR newborn piglets. CFTR localized tothe apical membrane component of crypt, but not villous cells in CFTR+/+tissues. No CFTR was detected in CFTR−/− tissues (FIG. 1E). CFTR wasvariably present in ileum from the different CFTR−/−; TgFABP-pCFTR linesand was most consistently detected in the lines with the favorableintestinal phenotype (FIG. 1E). In contrast to the villous celllocalization of human CFTR expressed under control of the rat iFABPpromoter (Zhou et al., Science 266:1705-1708, 1994) or villin promoter(Lu et al., Pediatr. Pulmonol. Suppl. 12:213, 1995) in mice, porcineCFTR localized to crypt cells in the CFTR−/−; TgFABP-pCFTR intestines(FIG. 1E). This finding is in agreement with other reports of endogenousCFTR localization in the intestinal crypt cells (Rogers et al., Science321:1837-1841, 2008; Odes et al., Acta Physiol. Scand. 178:231-240,2003; Trezise et al., Nature 353:434-437, 1991; Strong et al., J. Clin.Invest. 93:347-354, 1994).

Transgenic Expression of iFABP-pCFTR in CFTR−/− Pigs IncreasesIntestinal CFTR-Mediated CC Transport

We next asked if iFABP promoter driven expression of CFTR led tofunctional CFTR activity in the intestine. Excised ileal segments weremounted in Ussing chambers and transepithelial currents were assayed.Addition of forskolin and 3-isobutyl-2-methylxanthine (IBMX), toincrease cellular concentrations of cAMP and phosphorylate and activateCFTR, increased short-circuit current (Isc) in ileal segments of CFTR+/+intestine, but failed to induce a significant response in CFTR−/−intestine (FIGS. 2A and 2B).

Similar to the variable CFTR mRNA levels observed across the differentCFTR−/−; TgFABP-pCFTR cell lines, we also observed a range of forskolinand IBMX-stimulated Isc in ileum from transgenic animals. Lines 1a and1e had forskolin and IBMX responses in the range of wild-type tissue,while ileum from 1b and 1c failed to respond to forskolin and IBMX(similar to CFTR−/− tissue), and line 1d had an intermediate phenotype(FIGS. 2A-2D). Compared to the forskolin and IBMX response observed fromCFTR+/+ ileal tissues, the average ileal response in samples from lines1a and 1e were 63 and 84% of control tissue values, respectively. Theforskolin and IBMX Isc values in lines 1b and 1c were similar to CFTR−/−and line 1d was 27% of CFTR+/+(FIG. 2D and Table 1).

The forskolin and IBMX response in ileal segments correlated very wellwith the intestinal phenotype observed between the different CFTR−/−;TgFABP-pCFTR lines. For example, lines 1a and 1e had the greatestforskolin and IBMX Isc responses and the least severe intestinalphenotype. Furthermore, 27% of wild-type CFTR-mediated Cl⁻ transport inthe ileum is not sufficient to prevent meconium ileus, but 63% ofwild-type function does correct and/or alleviate the meconium ileusphenotype in CFTR−/− pigs (FIG. 2E).

Finally, in all of the lines studied, we observed a similar degree ofpancreatic destruction (FIGS. 3A-3B), micro-gallbladder (FIGS. 3C-3D),and focal biliary cirrhosis (FIG. 3E) as previously reported in CFTR−/−and CFTRΔF508/ΔF508 newborn piglets (Rogers et al., Science321:1837-1841, 2008; Stoltz et al., Sci. Transl. Med. 2:29ra31, 2010;Meyerholz et al., Am. J. Pathol. 176:1377-1389, 2010; Ostedgaard et al.,Sci. Transl. Med. 3:74ra24, 2011). These findings demonstrate that theFABP-pCFTR transgene did not correct the extra-intestinal CFmanifestations typically observed in the digestive system of newbornpiglets and humans with CF.

CFTR is not Present in the Airways of CFTR−/−; TgFABP-pCFTR Pigs

In mice, endogenous iFABP expression is localized to the intestinaltract. However, when hCFTR was expressed under control of the rat iFABPpromoter in CFTR-null mice, in certain lines some extra-intestinal hCFTRexpression was observed, including in the lungs (Zhou et al., Science266:1705-1708, 1994). Therefore, we next determined if CFTR was beingexpressed in the airways of CFTR−/−; TgFABP-pCFTR pigs. On grossexamination, CFTR−/−; TgFABP-pCFTR tracheas had narrowed airway lumensand cartilaginous ring defects as previously observed in neonatal pigsand humans with CF (Meyerholz et al., Am. J. Resp. Crit. Care. Med.182:1251-1261, 2010) (FIG. 4). These findings were present in all lines(lines 1a-1e) studied. Using immunostaining for CFTR in newborn pigletCFTR+/+ tracheas, we found that CFTR localized to the apical membranecomponent of airway epithelial cells. No CFTR was detected in CFTR−/− orCFTR−/−; TgFABP-pCFTR tracheas.

We next harvested fresh tracheal tissue from newborn piglets, mountedthe excised tissue in Ussing chambers, and measured transepithelialcurrents to confirm the absence of CFTR-mediated Cl⁻ currents inCFTR−/−; TgFABP-pCFTR tracheas. Tracheal tissues were obtained from atleast one piglet from each of the transgenic lines studied. Followingamiloride addition, to block epithelial Na⁺ channels, we added4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) to blocknon-CFTR Cl⁻ channels, followed by forskolin and IBMX treatment toassess CFTR function. Forskolin and IBMX treatment significantlyincreased Isc in CFTR+/+ tracheas, but failed to increase Isc in CFTR−/−or any of the tracheas from the different

CFTR−/−; TgFABP-pCFTR transgenic lines (FIGS. 5A-D). We performedsimilar measurements on cultured tracheal and nasal turbinate epitheliumfrom CFTR+/+, CFTR−/−, and CFTR−/−; TgFABP-pCFTR pigs and foundqualitatively very similar results to the excised tracheal studies(FIGS. 5E-F). These findings show that with this iFABP-pCFTR construct,we observe no CFTR expression in the proximal airways. These findingsare important because they suggested that the CFTR−/−; TgFABP-pCFTR pigsmight go on to develop lung disease similar to the CFTR−/− andCFTRΔF508/ΔF508 pigs (Stoltz et al., Sci. Transl. Med. 2:29ra31, 2010;Ostedgaard et al., Sci. Transl. Med. 3:74ra24, 2011).CFTR−/−; TgFABP-pCFTR Pigs Develop Lung Disease Similar to Humans withCF

Of the 21 piglets treated with enemas (from 5 different transgeniclines), the bowel obstruction was relieved in 5 animals (from lines 1aand 1e) and all of these animals lived greater than 5 days (Table 2). 2of these animals (cases #1 and #2) were euthanized at 6 days of age dueto sepsis. Microbiologic cultures were positive for Klebisellapneumoniae in both of these animals. In subsequent surviving piglets, wetreated animals with daily antibiotics for the first 7-14 days of lifeto minimize infectious complications.

TABLE 2 TRANSGENIC REASON FOR CASE SEX GENOTYPE LINE AGE EUTHANASIA 1Male CFTR−/−; 1a  6 d Sepsis TgFABP- (Klebsiella) pCFTR 2 Male CFTR−/−;1a  6 d Sepsis TgFABP- (Klebsiella) pCFTR 3 Male CFTR−/−; 1e  62 dGastric ulcer TgFABP- pCFTR 4 Male CFTR−/−; 1e 332 d Elective TgFABP-pCFTR 5 Male CFTR−/−; 1e 435 d Elective TgFABP- pCFTR

Our third surviving piglet (case #3) was euthanized at 62 days of agedue to signs of respiratory disease. The trachea was irregularly shaped,similar in appearance to newborn CF pig tracheas, and defects in thecartilage rings were present (Meyerholz et al., Am. J. Resp. Crit. Care.Med. 182:1251-1261, 2010) (FIG. 6A). Necropsy showed a large gastriculcer that had perforated into the left pleural space with evidence of apolymicrobial infection (gastrointestinal etiology) in the lungparenchyma, making interpretation of the CF-related lung diseasedifficult. Swine are particularly susceptible to gastric ulcers andthese have been linked to respiratory disease (Trobo et al., J. Med.25:251-254, 1994). We have previously reported a high incidence ofgastric ulcers in both CFTR−/− and CFTRΔF508/ΔF508 pigs (Stoltz et al.,Sci. Transl. Med. 2:29ra31, 2010; Ostedgaard et al., Sci. Transl. Med.3:74ra24, 2011). In addition to the gastric ulcer, the liver showedchanges of focal biliary cirrhosis (FIGS. 6C-E) and there wasfibroadipose tissue replacement of the pancreas with prominent dilatedducts and acini (FIG. 6B).

The two remaining CFTR−/−; TgFABP-pCFTR pigs were electively euthanizedat 332 (case #4) and 435 (case #5) days. Both animals had evidence ofprogressive loss of pancreatic exocrine tissue and replacement byadipose tissue, similar to older CFTR−/− and CFTRΔF508/ΔF508 pigs(Stoltz et al., Sci. Transl. Med. 2:29ra31, 2010; Ostedgaard et al.,Sci. Transl. Med. 3:74ra24, 2011) (FIGS. 7A-D). The gallbladders weresmall in size from case #4 and #5. The lumen of case #5's gallbladderwas filled with mucopurulent and necrotic cellular debris (FIGS. 7E-G)and the lumen of case #4 was also filled with mucus and inflammatorycells were present. We also observed variable changes along the lengthof the vas deferens in both case #4 and #5. In some areas of the vasdeferens, the lumen was collapsed and filled with eosinophilic materialthat was PAS-positive, but more commonly the lumen was completely absent(FIGS. 8A and 8B).

Both pigs had variable evidence of lung disease at necropsy and onserial chest CTs. Lung disease tended to be most evident in the tracheallobe and FIG. 9A shows serial chest CT images of the tracheal loberegion from case #5 at 61, 92, 148, 219, 260, and 308 days of age. Therewas evidence of progressive involvement of the tracheal lobe with airwaywall thickening, patchy infiltrates, and progressive collapse of asubsegment of the tracheal lobe. Over time some of these changes werevariably present, while others (airway wall thickening) seemed toremain.

Serial bronchoscopy with bronchoalveolar (BAL) lavage was performed onthese two animals to look for evidence of airway infection andinflammation. Culture of BAL liquid showed variable numbers of bacteriain samples from these two animals over time. In all but two of the BALliquid samples (both from case #5), at least 10² cfu/ml of totalbacteria was cultured (FIGS. 9B and 9D). A number of different bacterialspecies were cultured from the BAL liquid (FIGS. 9C and 9E). Of note,Pseudomonas aeruginosa was cultured from the BAL liquid of one pig (case#5) at three different time points (FIG. 9E) with the greatest amountbeing present on the first BAL. P. aeruginosa is a common bacterialpathogen isolated from the lungs of humans with CF and this is the firsttime that we have isolated P. aeruginosa from lungs of a CF pig (CFTR−/−or CFTRΔF508/ΔF508). Interestingly, the absolute number of P. aeruginosacultured from the BAL liquid changed over time and following the firstisolation of P. aeruginosa from BAL liquid samples, not all subsequentsamples were positive for P. aeruginosa; despite lack of antibiotic usefor eradication of the pathogen. Streptococcus suis, a commonrespiratory pathogen in swine, was also isolated in large numbers fromcase #5 on day 219 (FIG. 9E).

In one of the two older animals (case #5), we found that the percentageof neutrophils recovered from the BAL liquid increased on days 148, 219,and 260 (FIG. 9F). Interestingly, these levels correlated with worseningradiographic abnormalities on the chest CT, increasing BAL liquidbacterial counts, and elevated IL-8 levels (FIGS. 9A, 9D, and 9G).

Materials and Methods CFTR−/−; TgFABP-pCFTR Vector Construction

For generation of CFTR−/−; TgFABP-pCFTR pigs, we transfected fetalfibroblasts with a transgenic construct containing the rat iFABPpromoter (a gift from Jeffrey Gordon, Washington University, St. Louis,Mo.) and porcine CFTR. Using the pCI vector (Promega), we firstsubstituted the CMV promoter with the nucleotide sequence −1178 to +28of the rat iFABP promoter. The porcine CFTR sequence was amplified frompcDNA3.1-Kozak-pCFTR plasmid using NheI and Xhol and inserted into theredesigned pCI-iFABP vector with the same enzymes. PlasmidpcDNA3.1(+)Hygro (Invitrogen) was digested with NruI and EcoRV to removethe CMV promoter (pcDNA3.1(+)HygroACMV). The iFABP-CFTR fragment wasamplified from pCI-iFABP-pCFTR with PfuUltra Polymerase (Stratagene) andligated with pcDNA3.1(+)HygroACMV. Restriction and sequence analysis wasperformed on the redesigned plasmid prior to use for transfection offetal fibroblasts (FIG. 1E).

Fetal Fibroblasts

We previously reported generation of CFTR−/− pigs (Rogers et al.,Science 321:1837-1841, 2008). Fetal fibroblasts were isolated from day35 CFTR−/− fetuses as previously described. Cells were grown in F10media (Invitrogen), containing 20% FCS and penicillin/streptomycin.

Transfection, Selection, and Southern Blot Screening of Clones

Fetal CFTR−/− fibroblasts were electroporated with 15 μg of linearizedDNA from the iFABP-pCFTR transgenic construct and then cultured. After48 hours, the media was changed and supplemented with 100 μg/mlhygromycin (Invitrogen) for 14 days to select for antibiotic-resistantclones. Southern blot screening was performed on hygromycin-resistantcells as previously described (Rogers et al., J. Clin. Invest.118:1571-1577, 2008). Briefly, amplified whole genomic DNA was preparedfrom each clone and 20 μg was digested with Ban II overnight. Genomicdigests were electrophoresed on a 0.8% agarose gel and transferred to apositively charged nylon membrane (Roche) by using an alkaline transferprocedure. Blots were prehybridized for 30 min at 65° C. in Rapid-hybbuffer (Amersham). The blot was then hybridized in Rapid-hyb buffer witha ³²P-labeled probe specific for the entire exon 13 of porcine CFTR (65°C., overnight). Two bands were anticipated on the blots: one at 2198 bpfor endogenous CFTR and the other at 3542 bp for the transgenic CFTR.

Production of Transgenic Animals

Cryopreserved clones were thawed and resuspended in micromanipulationmedia. Oocyte maturation, somatic cell nuclear transfer, surrogatepreparation, and embryo transfer were all performed as previouslydescribed (Rogers et al., J. Clin. Invest. 118:1571-1577, 2008). TheUniversity of Iowa and University of Missouri Animal Care and UseCommittees approved all animal studies.

Care of Pigs

A cesarean section was performed to recover the piglets on days 116-118.After delivery the piglets were provided medical care and fed colostrumcontaining polyethylene glycol 3350. Shortly after birth, most pigletsunderwent a gastrograffin enema. This was repeated every 8-12 hoursuntil all meconium was evacuated or the enemas failed to induce aresponse. Piglets were initially raised on milk replacer until matureenough to be placed on standard pig diets. In some litters, pigletsreceived daily antibiotics for the first 7-14 days of life (ceftiofur, 5mg/kg, IM). All animals received: a) oral pancreatic enzyme replacementtherapy (PancreVed, Vedco) with meals (4000 IU lipase/120 mL milkreplacer or ˜10,000 IU lipase/kg/day divided between meals). b) oralfat-soluble vitamins (PancreVed, Vedco). c) oral proton pump inhibitor(1 mg/kg) (omeprazole, Sandoz) once a day. d) oral polyethylene glycol3350 (Paddock Laboratories, MinneapPharmaceuticals) with each mealtitrated to maintain soft stools.

Bronchoalveolar Lavage Liquid Collection and Analysis

For bronchoalveolar lavage (BAL) in the older pigs, animals wereanesthetized with ketamine (15-20 mg/kg, IM), xylazine (1-1.5 mg/kg,IM), and intravenous propofol. A flexible fiberoptic bronchoscope(Pentax FB-10X; Montvale, N.J.) was inserted orally and passed throughthe vocal cords. The suction channel was not used until the tip of thebronchoscope was past the vocal cords. The bronchoscope tip was gentlyinserted and wedged into the tracheal bronchus airway. Three aliquots(10 ml each) of sterile saline were instilled into the airway and lavageliquid was recovered with intermittent suction. The BAL liquid waspooled and immediately placed on ice, transported to the laboratory forprocessing (cell counts and microbiology studies) and stored at −80° C.for subsequent analysis.

The total number of recovered cells in BAL liquid was quantified with ahemacytometer and morphologic differentiation of cells was performed oncytospin preparations that were stained with Diff-Quick Stain kit(Baxter). Microbiologic studies were performed on collected BAL liquid,and IL-8 levels were determined on recovered supernatant aftercentrifugation (1600×g for 10 min) using a standard sandwich ELISA (R&DSystems).

CT Scanning

All chest X-ray computed tomographic (CT) imaging was performed onsedated, spontaneously breathing animals using a 64-slicehigh-resolution CT (HRCT) scanner (SOMATOM 64, Siemens, Malvern, Pa.).

Production of Primary Cultures of Differentiated Airway Epithelia

Epithelial cells were isolated from tracheas and nasal turbinates byenzymatic digestion, seeded onto permeable filter supports, and grown atthe air-liquid interface as previously described. Differentiatedepithelia were used at least 14 days after seeding.

Histopathological Analysis

At necropsy, pigs were examined for gross lesions and the findings weredocumented. Tissues were fixed in 10% neutral buffered formalin for48-96 hr. Tissues were then routinely processed, embedded, sectioned (4μm), and stained with hematoxylin and eosin (HE) for generalexamination.

Microbiologic Studies

Standard microbiologic techniques were utilized to identify and quantifybacteria present in bronchoalveolar lavage liquid samples. Samples wereserially diluted and plated onto blood agar (tryptic soy agar with sheepblood; Remel), Colombia colistin-nalidixic acid agar (Remel), Chocolateagar (Remel), Mannitol Salt Agar (Remel), MacConkey agar (Remel), andBurkholderia cepacia selective agar (Remel). Organisms were identifiedwith standard microbiological procedures. Some identifications wereconfirmed by API 20E or API 20NE (bioMérieux), Vitek (bioMérieux) or 16SrRNA gene sequencing (University of Iowa Clinical MicrobiologyLaboratory and Iowa State University Diagnostic Laboratory).

Quantitative RT-PCR

Quantitative RT-PCR used TaqMan chemistry and an ABI 7500 Fast Real-timePCR System to measure porcine CFTR mRNA. Briefly, tissue was collectedin RNAlater (Ambion) and total RNA was isolated as above. First-strandcDNA was synthesized with random hexamers (SuperScript III, Invitrogen).Sequence-specific primers and probes for porcine CFTR and b-actin werefrom ABI. For measuring CFTR mRNA, primer/probe sets annealing to exon10 of CFTR (Ss03389420_m1, pCFTR) and b-actin (Ss03376160_u1, ACTB) wereused in separate reactions. Amounts of CFTR mRNA were normalized toβ-actin. This normalized value for each tissue expressed relative tonormalized CFTR^(+/+) duodenum to calculate the % of mRNA relative towild-type duodenum (100%).

Immunoprecipitation and Phosphorylation

Protein assays were performed using the BCA assay (Thermo-Fisher).Indicated amounts of intestinal tissue or cells from 1-2 trachea werehomogenized in the Tris-mannitol buffer on ice with 20 strokes of theloose-fitting and 15 strokes of the tight-fitting pestle of aPotter-Elvejehm homogenizer. Membrane pellets were isolated bycentrifugation at 200,000×g at 4° C. for 30 min. The pellets weresolubilized in a commercial detergent mix, Membrane Solution 2(Profoldin, Ca). Soluble proteins were separated from insoluble pelletsby centrifuging at 200,000×g for 20 min. CFTR was immunoprecipitatedfrom the supernatant of soluble proteins with anti-CFTR antibodies M3A7and MM13-14 (Upstate Biotechnology) and in vitro phosphorylated with³²P-ATP and the catalytic subunit of PKA (Promega). Washed precipitateswere electrophoresed on 6% SDS-PAGE. Gels were stained, destained, driedand exposed to phosphoscreens before imaging on a Fuji FLA7000 imager(General Electric).

Immunocytochemistry

Intestinal and tracheal tissues were excised from newborn piglets,immediately placed in ice-cold 30% sucrose, and quick-frozen in OCT withliquid N₂. Tissue segments were kept at −80° C. Tissues werecryosectioned into 7 μm sections, fixed in 100% MeOH at −20° C. for 10min, permeabilized in 0.2% TX-100 (Thermo-Fisher) in PBS, and blocked inSuper-Block (Thermo-Fisher) with 5% normal goat serum (JacksonImmunoResearch). Tissue sections were incubated for 2 hrs at 37° C. inanti-CFTR antibodies MM13-4, M3A7 (Chemicon), and polyclonal antibody tothe tight junction protein ZO-1 (Zymed) (all at 1:100 dilution),followed by secondary antibodies (goat-anti-mouse Alexa-Fluor488 andgoat anti-rabbit Alexa-Fluor568; Molecular Probes/Invitrogen) (1:1000dilution). Sections were mounted with Vectashield (Hard-set) containingDAPI (Vector Labs) to visualize nuclei. Images were acquired withidentical parameters on an Olympus Fluoview FV 1000 confocal microscopewith a UPLSAPO 60× oil lens. Images were scanned sequentially at 2msec/pixel.

Electrophysiological Measurements of Freshly Excised and CulturedEpithelia

Epithelial tissues were excised from the nasal turbinate and tracheaimmediately after animals were euthanized. Tissues and culturedepithelia were studied in modified Ussing chambers. Epithelia werebathed on both surfaces with solution containing (mM): 135 NaCl, 2.4K₂HPO₄, 0.6 KH₂PO₄, 1.2 CaCl₂, 1.2 MgCl₂, 10 dextrose, 5 HEPES, pH 7.4,at 37° C. and gassed with compressed air. Transepithelial voltage (Vt)was maintained at 0 mV to measure short-circuit current (Isc).Transepithelial electrical conductance (Gt) was measured byintermittently clamping Vt to +5 and/or −5 mV.

A standard protocol was the following. 1) Measurements under basalconditions. 2) 100 mM apical amiloride to inhibit ENaC Na⁺ channels. 3)100 mM apical DIDS (4,4-diisothiocyano stilbene-2,2-disulfonic acid) toinhibit most anion channels other than CFTR. 4) 10 mM forskolin and 100mM IBMX (3-isobutyl-2-methylxanthine) to increase cellular levels ofcAMP leading to phosphorylation and activation of CFTR. 5) 100 mM apicalGlyH-101 to inhibit CFTR. 6) 100 μM bumetanide to inhibit basolateralNa—K-2Cl transporter in cultured epithelia.

OTHER EMBODIMENTS

All publications, patents, and other citations noted in thisspecification are incorporated herein by reference as if each individualpublication, patent, or other citation were specifically andindividually indicated to be incorporated by reference. Although theinvention has been described above in some detail by way of illustrationand example for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

Use in the claims and elsewhere herein of singular forms, such as “a”and “the,” does not exclude indication of the corresponding plural form,unless the context indicates to the contrary. Thus, for example, if aclaim indicates the presence of “a” mutation in “a” gene, it can beinterpreted as covering one or more mutations, in one or more genes,unless otherwise indicated. Further, the term “or” as used herein isintended to be interpreted as both optional (i.e., one or the other andnot both of multiple options) and inclusive (i.e., and/or).

Other embodiments are within the following claims.

1. A transgenic pig comprising a genome that comprises: (i) one or more mutations in both alleles of the pig cystic fibrosis transmembrane regulator (CFTR) gene resulting in altered pig CFTR expression and one or more symptoms of cystic fibrosis (CF) in the pig; and (ii) a CFTR transgene under the control of a promoter resulting in CFTR expression in intestinal cells of the pig; wherein said pig exhibits reduced meconium ileus relative to a pig lacking expression of said CFTR transgene in intestinal cells.
 2. The transgenic pig of claim 1, wherein the promoter is intestinal cell-specific.
 3. The transgenic pig of claim 2, wherein said intestinal cell-specific promoter is an intestinal fatty acid binding protein (iFABP) promoter.
 4. The transgenic pig of claim 1, wherein the CFTR alleles of the genome of said pig have been knocked out.
 5. The transgenic pig of claim 4, wherein the CFTR alleles of the genome of said pig have been replaced with mutant CFTR genes, the expression of which results in a CF phenotype.
 6. The transgenic pig of claim 5, wherein one or both of said mutant CFTR genes is a mutant human CFTR gene.
 7. The transgenic pig of claim 5, wherein one or both of said mutant CFTR genes is a mutant porcine CFTR gene.
 8. The transgenic pig of claim 5, wherein said mutant CFTR gene comprises a deletion of F508.
 9. The transgenic pig of claim 1, wherein the pig has one or more phenotypes selected from the group consisting of (a) an electrophysiological phenotype similar to that of human cystic fibrosis, (b) pancreatic insufficiency or abnormalities, (c) hepatic abnormalities, (d) gall bladder and/or bile duct abnormalities, (e) tracheal abnormalities, (f) cystic fibrosis lung disease, (g) sweat gland abnormalities, and (h) kidney abnormalities.
 10. A method of determining whether a candidate therapeutic approach can be used in the treatment of cystic fibrosis, the method comprising carrying out said candidate therapeutic approach on a transgenic pig of claim 1 and monitoring the pig for one or more symptoms of cystic fibrosis, wherein detection of improvement in one or more symptoms of cystic fibrosis indicates the identification of a therapeutic approach that can be used in the treatment of cystic fibrosis.
 11. The method of claim 10, wherein the candidate therapeutic approach comprises administration of a candidate therapeutic agent.
 12. The method of claim 10, wherein the symptom of cystic fibrosis is monitored in the lung, pancreas, liver, or kidney of the pig.
 13. An isolated cell or tissue of a pig of claim
 1. 14. A heterozygous form of a transgenic pig of claim
 1. 15. An isolated cell or tissue of the transgenic pig of claim
 14. 